Composition for measuring glucose having improved substrate specificity

Abstract

The present invention relates to a method for lowering activity with respect to maltose in glucose measurement comprising a step of reacting modified pyrroloquinoline quinone dependent glucose dehydrogenase subjected to amino acid sequence modification, wherein pyrroloquinoline quinone dependent glucose dehydrogenase is reacted in the presence of at least one type of substance selected from the group comprising succinic acid, malonic acid, glutaric acid, malic acid, phthalic acid, 2-ketoglutaric acid, 3,3-dimethylglutaric acid, pimeric acid, suberic acid, adipic acid, maleic acid, potassium chloride, ammonium chloride, diammonium hydrogen citrate, L-lysine, taurine, calcium glycerate, amino-n-butyric acid, sodium glycolate, sodium α-ketoglutarate, fumaric acid, glycine, glutamic acid, serine and citric acid.

1. A method for lowering a reactivity on maltose in glucose measurement comprising a step of reacting modified pyrroloquinoline quinone dependent glucose dehydrogenase in the presence of at least one substance selected from the group consisting of succinic acid, malonic acid, glutaric acid, malic acid, phthalic acid, 2-ketoglutaric acid, 3,3-dimethylglutaric acid, pimeric acid, suberic acid, adipic acid, maleic acid, potassium chloride, ammonium chloride, diammonium hydrogen citrate, L-lysine, taurine, calcium glycerate, amino-n-butyric acid, sodium glycolate, sodium α-ketoglutarate, fumaric acid, glycine, glutamic acid, serine and citric acid, wherein the modified pyrroloquinoline quinone dependent glucose dehydrogenase consists of SEQ ID NO: 1 except that position 168 of SEQ ID NO: 1 and at least one position selected from the group consisting of positions 169, 170, 245, 342, and 351 of SEQ ID NO: 1 are substituted with another amino acid, and wherein a pH of 5.0 to 6.0 is maintained during the glucose measurement reaction. 
     
     
       2. The method according to  claim 1 , wherein the at least one substance is selected from the group consisting of succinic acid, malonic acid, glutaric acid, malic acid, phthalic acid, 2-ketoglutaric acid, 3,3-dimethylglutaric acid, pimeric acid, suberic acid, adipic acid, maleic acid and citric acid. 
     
     
       3. The method according to  claim 1 , wherein the at least one substance is selected from the group consisting of succinic acid, adipic acid, suberic acid, pimeric acid, potassium chloride, ammonium chloride, diammonium hydrogen citrate, malonic acid, L-lysine, taurine, 3,3-dimethylglutaric acid, malic acid, glutaric acid, calcium glycerate, amino-n-butyric acid, sodium glycolate, sodium α-ketoglutarate, fumaric acid, glycine, glutamic acid and serine. 
     
     
       4. The method for lowering a reactivity on maltose in the measurement of glucose according to  claim 1 , wherein the total added amount of at least one substance selected from the group consisting of succinic acid, malonic acid, glutaric acid, malic acid, phthalic acid, 2-ketoglutaric acid, 3,3-dimethylglutaric acid, pimeric acid, suberic acid, adipic acid, maleic acid, potassium chloride, ammonium chloride, diammonium hydrogen citrate, L-lysine, taurine, calcium glycerate, amino-n-butyric acid, sodium glycolate, sodium α-ketoglutarate, fumaric acid, glycine, glutamic acid, serine and citric acid is 0.05% or more as a percent by weight in solution. 
     
     
       5. The method according to  claim 1 , wherein the reaction is carried out in the presence of at least one mediator. 
     
     
       6. The method according to  claim 5 , wherein the mediator is a ferricyanide salt. 
     
     
       7. The method according to  claim 1 , wherein the modified pyrroloquinoline quinone dependent glucose dehydrogenase has a lowered reactivity on maltose as compared with an enzyme having SEQ ID NO: 1. 
     
     
       8. A composition for measuring glucose comprising (a) a modified pyrroloquinoline quinone dependent glucose dehydrogenase consisting of SEQ ID NO: 1 except that position 168 of SEQ ID NO: 1 and at least one position selected from the group consisting of positions 169, 170, 245, 342, and 351 of SEQ ID NO: 1 are substituted with another amino acid, and (b) one or more substances selected from the group consisting of succinic acid, malonic acid, glutaric acid, malic acid, phthalic acid, 2-ketoglutaric acid, 3,3-dimethylglutaric acid, pimeric acid, suberic acid, adipic acid, maleic acid, potassium chloride, ammonium chloride, diammonium hydrogen citrate, L-lysine, taurine, calcium glycerate, amino-n-butyric acid, sodium glycolate, sodium α-ketoglutarate, fumaric acid, glycine, glutamic acid, serine and citric acid. 
     
     
       9. A glucose sensor comprising (a) a modified pyrroloquinoline quinone dependent glucose dehydrogenase consisting of SEQ ID NO: 1 except that position 168 of SEQ ID NO: 1 and at least one position selected from the group consisting of positions 169, 170, 245, 342, and 351 of SEQ ID NO: 1 are substituted with another amino acid, and (b) one or more substances selected from the group consisting of succinic acid, malonic acid, glutaric acid, malic acid, phthalic acid, 2-ketoglutaric acid, 3,3-dimethylglutaric acid, pimeric acid, suberic acid, adipic acid, maleic acid, potassium chloride, ammonium chloride, diammonium hydrogen citrate, L-lysine, taurine, calcium glycerate, amino-n-butyric acid, sodium glycolate, sodium α-ketoglutarate, fumaric acid, glycine, glutamic acid, serine and citric acid. 
     
     
       10. A kit for measuring glucose comprising the composition of  claim 8 . 
     
     
       11. A method for measuring glucose comprising using the glucose sensor of  claim 9 , to measure glucose of a sample.